Fluorescence is a wide-spread technique in biology to investigate several problems, ranging from cellular and in vivo imaging to molecular diagnostics. The technique is based on labelling particles of interest with fluorophores and on detecting the labelled particles by irradiating them with an excitation source and detecting the fluorescent emission. In molecular diagnostics, fluorescence is often used to determine the concentration of an analyte, i.e., the particles of interest. Although this is a very sensitive technique, it usually requires expensive and bulky free-space optics.
Several efforts have been made to miniaturize fluorescence detection setups. Miniaturisation has been obtained by integrating the detector into the sensing element. Fluorescence is generated by optical excitation of fluorescent particles that typically are immobilized on the surface of the sensing element. The subsequent emission of fluorescence by these particles is then detected by a detector integrated in the sensing element. In order to efficiently excite the fluorescent particles, this involves typically a strong radiation source (usually a laser source), that has a shorter wavelength than the wavelength of the fluorescence emission. The optical power in the excitation source is orders of magnitude larger than the emission from the molecules. Therefore, the excitation wavelength has to be strongly rejected, in order not to obscure the emitted radiation or even in order not to prevent detection from being possible, e.g. through saturation of the detector.
In existing, free-space, fluorescence setups, this is accomplished by making use of free-space optical elements such as a dichroic mirror and interference filters. Such elements are readily available and have proven efficiency. Nevertheless, when the detector is integrated in the sensing element, such filters need to be integrated in the device. For an integrated solution, such a filter needs to be introduced between the sensor surface, where the fluorescence particles are immobilized and excited, and the detector where the fluorescence detection needs to be detected. For very low emission detection, e.g., for the detection of very low concentrations, the rejection has to be very strong. Rejection ratios of 6 to 8 orders of magnitude, thus corresponding with optical density OD6-OD8, may be required, which sets very large constraints on the excitation radiation filter.
Some solutions have already been investigated. In one solution, interference filters were used as rejection filter for rejecting excitation radiation from the detector. Nevertheless, for reaching rejection ratios in the order of OD6 to OD8 rejection, the amount of layers in the interference filter need to be high and the thicknesses of the layers to be used is very small. Manufacturing of such interference filters is typically e complex and/or time consuming.
Another solution is the use of absorption based filters. Nevertheless, such filters typically suffer from heat generation in the filter, which may result in deterioration of the rejection properties as well as on the overall sensing quality of the sensor. Furthermore, it may be difficult to find an appropriate absorption material that absorbs at the proper excitation wavelength and that is transparent for the emission of the fluorescence particles of the most commonly used dyes.